The de novo purine biosynthetic pathway generates inosine monophosphate, the precursor to purines and their derivatives that act in numerous cellular anabolic and catabolic networks. The pathway consists of enzymes that catalyze ten chemical steps and, except for being mono- or multifunctional, are highly conserved from pro- to eukaryotes. Although the enzymes have long been hypothesized to exist in a multienzyme complex, only recently have we been able to demonstrate their clustering in vivo to form the purinosome. We propose to investigate the protein components of this cluster in terms of their stoichiometry and the possible inclusion of non-pathway proteins; of their protein-protein proximity within the purinosome; of putative post-translational modifications that drive purinosome assembly, and of their effect when clustered on the formation of pathway generated metabolite levels. Detection, identification, and quantification of the purinosome associated enzymes and non-pathway proteins will make extensive use of cellular imaging of chimeric fluorescent protein constructs, of co- immunoprecipitation, and of blue native polyacrylamide gel electrophoresis, the latter two linked to MALDI/MS/MS. Tandem MS/MS will be the method of choice for investigations of putative post- translational modifications. The locus and stoichiometry of purinosome protein members within the complex will be sought by in vivo fluorescence resonance energy transfer (FRET), stochastic optical reconstruction microscopy (STORM), and intracellular reporter assays. The functional advantage of the purinosome to affect metabolite levels within cells will be probed by MALDI/MS/MS as well as secondary ion mass spectrometry (SIMS) and correlated with purinosome cluster density. Many of the individual enzymes, particularly those that utilize folate cofactors, have been biologically validated as chemotherapeutic targets. The proposed studies in the long term have the potential to expand greatly our understanding of how biosynthetic or catabolic pathways may organize intracellularly and the biochemical advantages to the cell of such multienzyme clusters as our approach is extended to other pathways.